Objective: To evaluate the impact of different diluting media on determination of serum estradiol (E₂) concentration by using chemiluminescent immunoassay. Methods: Fifty serum samples were collected for E₂ detection using chemiluminescent immunoassay by an automatic biochemical analyzer (UniCel DXI 800, Beckman Coulter). Four diluting media were used, including standard diluent S₀ (Beckman Coulter), pooled low-level E₂ serum (PS), E₂/progesterone standard diluent (Roche), and calf serum (CS). Samples were manually diluted into 1∶1 or 1∶4 using the above 4 media with two parallel tubes and detected by UniCel DXI 800. Multiple statistical methods were applied to analyze the data. Results: Students't test revealed that the E₂ concentration measured by diluting media CS, Roche and PS compared with S₀ showed significant differences statistically (P=5.03×10^-11, 1.44×10^-10 and 2.79×10^-13, respectively). Pearson correlation analysis showed that the correlation coefficient r between diluting media S₀ and CS, Roche, PS were 0.992 3, 0.971 3, 0.968 0, respectively. After correction with linear regression model, the concentration measured by diluting media CS, Roche and PS compared with S₀ showed no significant difference statistically (P=0.998 3, 0.997 9, 0.987 7, respectively). Consistence of E₂ concentration measured by diluting media CS, Roche and PS compared with S₀ was evaluated by Bland-Altman method, the results showed that 2%, 2% and 8% of the samples exceeded the consistence limit, respectively. The maximum bias of three assessed diluting media were 16.8%, 44.8% and 45.2%, respectively. CS shows the best consistence. Conclusions: Among the three assessed diluting media, CS shows the best performance and could serve as a replacement of manufacturer's standard diluent with high stability and lower cost.